Tryptamine, 5-Hydroxy- anticorps

Détails pour le produit réf. ABIN6559967
Immunocytochemistry (ICC)
Immunogène Synthetic 5-Hydroxytryptamine conjugated to protein carrier (Pc)
Isotype IgG
Specificité Conjugated 5-Hydroxytryptamine . Using a conjugate 5-Hydroxytryptamine-(Pc), antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) 5-Hydroxytryptamine-G-(Pc) 1 5-Methoxytryptamine-G-(Pc) 1/25 Tryptamine-G-(Pc) 1/50 5-Hydroxytryptophan-G-(Pc) 1/175 5-Hydroxytryptamine 1/1,150 L-Tryptophan-G-(Pc) 1/3,500 5-Methoxytryptophan-G-(Pc) 1/20,000 (a): 5-Hydroxytryptamine-G-(Pc) concentration / Other c conjugated indolealkylamines concentration at half displacement G = Glutaraldehyde
Purification Antiserum previously preabsorbed on protein carriers, and purified
Autre désignation Tryptamine, 5-Hydroxy
Classe de substances Chemical
Indications d'application Example of Immunocytochemistry application Detection of conjugated 5-Hydroxytryptamine (serotonin) in rat brain 1- Perfusion: The rat is anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the following solutions: Solution A: cacodylate 0.1M, sodium metabisulfite 10g/l, pH = 6.2 Solution B: cacodylate 0.1M, sodium metabisulfite 10g/l and glutaraldehyde 3-5%, pH = 7.5 2- Post fixation: 15 to 30 min in solution B, then 4 soft washes in Tris 0.05M with sodium metabisulfite 8.5g/l, pH 7.5 (solution C). 3- Tissue sectioning: Cryostat or vibratome sections can be used. 4- Reduction step: Sections are reduced with the solution C containing sodium borohydride (0.1M) for 10 min. Then, the sections are washed 4 times with solution C without sodium borohydride. 5- Application of anti-conjugated 5-Hydroxytryptamine antibodies: The final dilution is 1/1,000 to 1/5,000 in solution C containing triton X100 0.5%, plus 2% of non-specific serum. A dozen of sections can be incubated with 2ml of antibody solution overnight at 4°C. Then, after this period, the sections are washed 3 times (10 min) with solution C. N.B.: Antibodies may be used at a higher dilution. The customer should explore the antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. 6- PAP procedure: Second antibody: Sections are incubated with 1/100 dilution of goat anti-rabbit in solution C for 3 hours at 20°C or 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C PAP: Sections are incubated with 1/1000 dilution of rabbit peroxidase anti-peroxidase complex in solution C for 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C Revelation: Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated 0.05% of H2O2 is added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M. Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)
Restrictions For Research Use only
Format Lyophilized
Stock 4 °C